This relates to the fields of immunology and protein biochemistry and more particularly relates to RNA polymerase II peptides useful for nuclear targeting.
The notion that genes might be replaced or specifically inhibited seems realistic given the recent explosion in human genome research; the spectacular successes in transgenic animal technology; the development of viral and non-viral ex vivo gene transfer techniques; and the intermittent successes of antisense oligodeoxynucleotide and ribozyme technologies. The gene therapy concept has led to the formation of many biotech companies specializing in gene transfer, antisense oligonucleotides and catalytic RNAs. The pharmaceutical industry and the NIH have also made significant financial commitments to develop these technologies. Millions of dollars of investment capital and grant funds have been spent on gene therapy research, and several human gene therapy clinical trials are underway. Despite all of this excitement, genetic therapy is still in a very early stage of development. It is clear that some very difficult engineering problems must be overcome before gene transfer, antisense and catalytic RNA technologies will be used to treat human diseases.
Technical barriers can be divided into three categories: Reagent design: optimization of nucleic acid activity through gene transfer and antisense oligodeoxynucleotides and catalytic DNAs; Delivery: formulating and delivering nucleic acids into cells; and Targeting: ensuring that nucleic acid reagents are bioavailable after they enter the cell. Some of the issues that must be addressed in targeting and delivery include: a substantial fraction of the reagent (transgene/oligo/catalytic RNA) must enter the nucleus; intranuclear targeting of nucleic acid reagents must be optimized and intranuclear sequestration must be avoided; if stable expression is desired, transgenes must recombine with chromosomes; stable and transient transgenes must be accessible to the transcription machinery; and oligos and catalytic RNAs must gain access to their pre-mRNA targets.
Gene therapy research has emphasized reagent design and delivery, but not targeting. To tackle the problems of reagent design and delivery, the gene therapy researchers have drawn from a vast body of research on gene regulation, nucleic acid biochemistry, virology and membrane biology. Significant advances have been made in the understanding of genomic organization and chromatin structure; RNA polymerase II-mediated transcription; packaging and splicing of pre-mRNA; stability and translational efficiency of mRNA; kinetics and thermodynamics of nucleic acid hybridization; catalytic RNAs; viral `vectorology` and liposome-mediated transfer of nucleic acids across cell membranes. Based upon this strong fund of knowledge, some of the problems associated with reagent design and delivery have been successfully addressed. The problem of targeting--i.e. concentrating the reagent in the appropriate subnuclear compartment--has received far less attention. One reason may be that reagent design and delivery are perceived as more tractable problems. It seems relatively straightforward to increase the Tm of an oligonucleotide; to optimize the composition of a liposome; or to modify the enhancer in a plasmid, changes which can be rapidly assessed in vitro. On the other hand, it seems difficult to manipulate or even to investigate the fate of plasmids, antisense oligos or catalytic RNAs after they cross the plasma cell membrane.
It is not known if nucleic acid reagents are bioavailable or sequestered once they enter the nucleus. Some nucleic acid reagents. for example, plasmids and oligonucleotides, accumulate in the nucleus without the help of a nuclear localization signal (NLS). This is usually considered a fortunate outcome, since the reagent has entered the desired subcellular compartment. Furthermore, it is tempting to think that nuclear accumulation indicates bioavailability. However, one cannot know how much of a nucleic acid reagent is bioavailable, and how much is sequestered inside the nucleus. To consider whether a nucleic acid reagent actually gains access to the desired subnuclear compartment, one should ask why transfected oligonucleotides typically accumulate in the nucleus. Like small peptides, oligos do not require a nuclear localization sequence (NLS) to enter the nucleus, since they can passively traverse nuclear pores. Presumably, oligos that diffuse into the nucleus are rapidly bound to intranuclear macromolecules, which retain them in this organelle. The nucleus contains a huge surplus of DNA and RNA binding proteins. The majority of nucleic acid binding proteins (e.g. hnRNPs and DNA binding proteins) are not associated with chromosomes at a given time, and therefore have the potential to bind RNA and/or DNA oligonucleotides. Indeed, the nucleus may be viewed as a high capacity chromatographic column, packed with nucleic acid binding proteins. Adventitious binding interactions with such proteins may severely inhibit the activities of antisense DNA oligos, catalytic RNAs and Plasmids may not be free to recombine with the chromosomes, or they may be sequestered from the RNA polymerase II transcriptional machinery. Ironically, therefore, the accumulation of a nucleic acid reagent in the nucleus may reflect its sequestration rather than its bioavailability.
To develop effective non-viral gene transfer and oligonucleotide-based therapies, it is important to realize that the nucleus is densely packed with chromosomal and extrachromosomal nucleoprotein complexes which exist in a relatively insoluble therapeutic agents can diffuse freely throughout this organelle. Indeed, large segments of pre-mRNA molecules are probably `buried` within ribonucleoprotein complexes, so it may be very difficult for soluble oligonucleotides and ribozymes to gain access to their target nucleic acid sequences in vivo.
It is therefore an object of the present invention to provide a therapeutic tool for the delivery of therapeutic agents from the surface of a cell to the cell nucleus or from a cell receptor to a targeted gene, and in particular to target therapeutics to specific intranuclear regions of the cells where they are bioavailable.